Hydrophilic filtration during manufacture of vaccine adjuvants

ABSTRACT

An improved method for the manufacture of an oil-in-water emulsion involves three procedures: (i) preparation of a preliminary emulsion; (ii) micro fluidization of the preliminary emulsion to reduce its droplet size; and (iii) filtration of the microfluidized emulsion through a hydrophilic membrane. The emulsions are useful as vaccine adjuvants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase patent application ofPCT/IB2010/003273, filed Dec. 3, 2010, which claims priority to U.S.provisional patent application Ser. No. 61/283,517, filed Dec. 3, 2009,all of which are hereby incorporated by reference in the presentdisclosure in their entirety.

TECHNICAL FIELD

This invention is in the field of manufacturing oil-in-water emulsionadjuvants for vaccines, for example by microfluidization.

BACKGROUND ART

The vaccine adjuvant known as ‘MF59’ [1-3] is a submicron oil-in-wateremulsion of squalene, polysorbate 80 (also known as Tween 80), andsorbitan trioleate (also known as Span 85). It may also include citrateions e.g. 10 mM sodium citrate buffer. The composition of the emulsionby volume can be about 5% squalene, about 0.5% Tween 80 and about 0.5%Span 85. The adjuvant and its production are described in more detail inChapter 10 of reference 4, chapter 12 of reference 5 and chapter 19 ofreference 6.

As described in reference 7, MF59 is manufactured on a commercial scaleby dispersing Span 85 in the squalene phase and Tween 80 in the aqueousphase, followed by high-speed mixing to form a coarse emulsion. Thiscoarse emulsion is then passed repeatedly through a microfluidizer toproduce an emulsion having a uniform oil droplet size. As described inreference 6, the microfluidized emulsion is then filtered through a 0.22μm membrane in order to remove any large oil droplets, and the meandroplet size of the resulting emulsion remains unchanged for at least 3years at 4° C. The squalene content of the final emulsion can bemeasured as described in reference 8.

Various documents (e.g. references 9-12) disclose that MF59 can bemanufactured by microfluidization followed by filter sterilizationthrough a 0.22 μm polysulfone filter. Polysulfones are polymerscontaining sulfone groups (SO₂) in the main polymer backbone andpolysulfone filters are well known hydrophobic filters.

It is an object of the invention to provide further and improved methodsfor the production of microfluidized oil-in-water emulsions (such asMF59), in particular methods that are suitable for use on a commercialscale and which use filtration.

DISCLOSURE OF THE INVENTION

The invention provides a method for the manufacture of an oil-in-wateremulsion comprising squalene, wherein the method comprises steps of (i)preparation of a first emulsion having a first average oil droplet size,also known as a preliminary emulsion or a pre-emulsion; (ii)microfluidization of the first emulsion to form a second emulsion havinga second average oil droplet size which is less than the first averageoil droplet size; and (iii) filtration of the second emulsion using ahydrophilic membrane.

The invention also provides a method for the manufacture of anoil-in-water emulsion comprising steps of: (i) preparation of a firstemulsion having a first average oil droplet size of 5000 nm or less,also known as a preliminary emulsion or a pre-emulsion; (ii)microfluidization of the first emulsion to form a second emulsion havinga second average oil droplet size which is less than the first averageoil droplet size; and (iii) filtration of the second emulsion using ahydrophilic membrane.

The invention also provides a method for the manufacture of anoil-in-water emulsion comprising steps of (i) preparation of a firstemulsion having a first average oil droplet size, also known as apreliminary emulsion or a pre-emulsion; (ii) microfluidization of thefirst emulsion to form a second emulsion having a second average oildroplet size which is less than the first average oil droplet size; and(iii) filtration of the second emulsion using a hydrophilicpolyethersulfone membrane.

Optionally, the first emulsion may be prepared using a homogenizer asdescribed below.

As described in more detail below, the first emulsion may have anaverage oil droplet size of 5000 nm or less e.g. an average size between300 nm and 800 nm. The number of oil droplets in the first emulsion witha size >1.2 μm may be 5×10¹¹/ml or less, as described below. Oildroplets with a size >1.2 μm are disadvantageous as they can causeinstability of the emulsion due to agglomeration and coalescence ofdroplets [14].

After formation, the first emulsion may then be subjected to at leastone pass of microfluidization to form the second emulsion having areduced average oil droplet size. As described below, the average oildroplet size of the second emulsion is 500 nm or less. The number of oildroplets in the second emulsion having a size >1.2 μm may be 5×10¹⁰/mlor less, as described below. To achieve these characteristics it may benecessary to pass the emulsion components through the microfluidizationdevice a plurality of times, e.g. 2, 3, 4, 5, 6, 7 times.

The second emulsion may then be filtered through a hydrophilic membrane,e.g. a hydrophilic polyethersulfone membrane, to give an oil-in-wateremulsion that may be suitable for use as a vaccine adjuvant. The averageoil droplet size of the oil-in-water emulsion produced after filtrationmay be 220 μm or less, e.g. between 135-175 nm, between 145-165 nm, orabout 155 nm. The number of oil droplets having a size >1.2 μm presentin the oil-in-water emulsion produced after filtration may be 5×10⁸/mlor less, e.g. 5×10⁷/ml or less, 5×10⁶/ml or less, 2×10⁶/ml or less or5×10⁵/ml or less.

The final oil-in-water emulsion formed after filtration may have atleast 10² times fewer oil droplets having a size >1.2 μm in comparisonto the first emulsion, and ideally at least 10³ times fewer (e.g. 10⁴times fewer).

In some embodiments, more than one cycle of steps (i) and (ii) is usedprior to step (iii). Similarly, multiple repeats of individual steps (i)and (ii) may be used.

The invention can also be used for preparing oil-in-water emulsionadjuvants without utilizing microfluidisation e.g. for methods whichrely on alternative techniques such as thermoreversibility [40]. Inthese embodiments the invention provides a method for the manufacture ofan oil-in-water emulsion comprising squalene, wherein the methodcomprises steps of: (i) preparation of an oil-in-water emulsion havingan average oil droplet size below 1 μm (e.g. below 500 nm, below 250 nm,below 200 nm, below 150 nm, below 100 nm); and (ii) filtration of theemulsion using a hydrophilic membrane. Thus these embodiments takeadvantage of the hydrophilic membrane's suitability for preparingsqualene-containing oil-in-water emulsion adjuvants but do not requirethe use of microfluidisation beforehand.

Similarly, the invention provides the use of a hydrophilic membrane forpreparing an oil-in-water emulsion adjuvant. Further features of themembrane and the emulsion adjuvant are discussed below.

In general, methods of the invention are performed between 20-60° C.,and ideally at 40±5° C. Although the first and second emulsioncomponents may be relatively stable even at higher temperatures, thermalbreakdown of some components can still occur and so lower temperaturesare preferred.

Emulsion Components

The average oil droplet size (i.e. the number average diameter of theemulsion's oil droplets) may be measured using a dynamic lightscattering technique, as described in reference 13. An example of adynamic light scattering measurement machine is the Nicomp 380 SubmicronParticle Size Analyzer (from Particle Sizing Systems).

The number of particles having a size >1.2 μm may be measured using aparticle counter such as the Accusizer™ 770 (from Particle SizingSystems).

Methods of the invention are used for the manufacture of oil-in-wateremulsions. These emulsions include three core ingredients: an oil; anaqueous component; and a surfactant.

Because the emulsions are intended for pharmaceutical use then the oilwill typically be biodegradable (metabolizable) and biocompatible.

The oil used may comprise squalene, a shark liver oil which is abranched, unsaturated terpenoid (C₃₀H₅₀;[(CH₃)₂C[═CHCH₂CH₂C(CH₃)]₂═CHCH₂—]₂;2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN7683-64-9). Squalene is particularly preferred for use in the presentinvention.

The oil of the present invention may comprise a mixture (or combination)of oils e.g. comprising squalene and at least one further oil.

Rather than (or on addition to) using squalene an emulsion can compriseoil(s) including those from, for example, an animal (such as fish) or avegetable source. Sources for vegetable oils include nuts, seeds andgrains. Peanut oil, soybean oil, coconut oil, and olive oil, the mostcommonly available, exemplify the nut oils. Jojoba oil can be used e.g.obtained from the jojoba bean. Seed oils include safflower oil,cottonseed oil, sunflower seed oil, sesame seed oil and the like. In thegrain group, corn oil is the most readily available, but the oil ofother cereal grains such as wheat, oats, rye, rice, teff, triticale andthe like may also be used. 6-10 carbon fatty acid esters of glycerol and1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and so may be used. The procedures forseparation, purification, saponification and other means necessary forobtaining pure oils from animal sources are well known in the art.

Most fish contain metabolizable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Squalane, the saturated analog to squalene, can also be used. Fish oils,including squalene and squalane, are readily available from commercialsources or may be obtained by methods known in the art.

Other useful oils are the tocopherols, particularly in combination withsqualene. Where the oil phase of an emulsion includes a tocopherol, anyof the α, β, γ, δ, ε or ξ tocopherols can be used, but α-tocopherols arepreferred. D-α-tocopherol and DL-α-tocopherol can both be used. Apreferred α-tocopherol is DL-α-tocopherol. The tocopherol can takeseveral forms e.g. different salts and/or isomers. Salts include organicsalts, such as succinate, acetate, nicotinate, etc. If a salt of thistocopherol is to be used, the preferred salt is the succinate. An oilcombination comprising squalene and a tocopherol (e.g. DL-α-tocopherol)can be used.

The aqueous component can be plain water (e.g. w.f.i.) or can includefurther components e.g. solutes. For instance, it may include salts toform a buffer e.g. citrate or phosphate salts, such as sodium salts.Typical buffers include: a phosphate buffer; a Tris buffer; a boratebuffer; a succinate buffer; a histidine buffer; or a citrate buffer.Buffers will typically be included in the 5-20 mM range.

The surfactant is preferably biodegradable (metabolizable) andbiocompatible. Surfactants can be classified by their ‘HLB’(hydrophile/lipophile balance), where a HLB in the range 1-10 generallymeans that the surfactant is more soluble in oil than in water, and aHLB in the range 10-20 are more soluble in water than in oil. Emulsionspreferably comprise at least one surfactant that has a HLB of at least10 e.g. at least 15, or preferably at least 16.

The invention can be used with surfactants including, but not limitedto: the polyoxyethylene sorbitan esters surfactants (commonly referredto as the Tweens), especially polysorbate 20 and polysorbate 80;copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butyleneoxide (BO), sold under the DOWFAX™ tradename, such as linear EO/PO blockcopolymers; octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); polyoxyethylene fatty ethersderived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brijsurfactants), such as triethyleneglycol monolauryl ether (Brij 30);polyoxyethylene-9-lauryl ether; and sorbitan esters (commonly known asthe SPANs), such as sorbitan trioleate (Span 85) and sorbitanmonolaurate. Preferred surfactants for including in the emulsion arepolysorbate 80 (Tween 80; polyoxyethylene sorbitan monooleate), Span 85(sorbitan trioleate), lecithin and Triton X-100.

Mixtures of surfactants can be included in the emulsion e.g. Tween80/Span 85 mixtures, or Tween 80/Triton-X100 mixtures. A combination ofa polyoxyethylene sorbitan ester such as polyoxyethylene sorbitanmonooleate (Tween 80) and an octoxynol such ast-octylphenoxy-polyethoxyethanol (Triton X-100) is also suitable.Another useful combination comprises laureth 9 plus a polyoxyethylenesorbitan ester and/or an octoxynol. Useful mixtures can comprise asurfactant with a HLB value in the range of 10-20 (e.g. Tween 80, with aHLB of 15.0) and a surfactant with a HLB value in the range of 1-10(e.g. Span 85, with a HLB of 1.8).

Formation of the First Emulsion

Before the microfluidization step, emulsion components may be mixed toform a first emulsion.

Oil droplets in the first emulsion may have an average size of 5000 nmor less e.g. 4000 nm or less, 3000 nm or less, 2000 nm or less, 1200 nmor less, 1000 nm or less, e.g. an average size between 800 and 1200 nmor between 300 nm and 800 nm.

In the first emulsion the number of oil droplets with a size >1.2 μm maybe 5×10¹¹/ml or less, e.g. 5×10¹⁰/ml or less or 5×10⁹/ml or less.

The first emulsion may then be microfluidised to form a second emulsionhaving a lower average oil droplet size than the first emulsion and/orfewer oil droplets with size >1.2 μm.

The average oil droplet size of the first emulsion can be achieved bymixing the first emulsion's components in a homogenizer. For instance,as shown in FIG. 1, they can be combined in a mixing vessel (12) andthen the combined components can be introduced (13) into a mechanicalhomogenizer, such as a rotor-stator homogenizer (1).

Homogenizers can operate in a vertical and/or horizontal manner. Forconvenience in a commercial setting, in-line homogenizers are preferred.

The components are introduced into a rotor-stator homogenizer and meet arapidly rotating rotor containing slots or holes. The components arecentrifugally thrown outwards in a pump like fashion and pass throughthe slots/holes. In some embodiments the homogenizer includes multiplecombinations of rotors and stators e.g. a concentric arrangement ofcomb-teeth rings, as shown by features (3) & (4); (5) & (6) and (7) &(8) in FIG. 1 and by FIG. 2. The rotors in useful large-scalehomogenizers may have comb-teeth rings on the edge of a horizontallyoriented multi-bladed impeller (e.g. feature (9) in FIG. 1) aligned inclose tolerance to matching teeth in a static liner. The first emulsionforms via a combination of turbulence, cavitation and mechanicalshearing occurring within the gap between rotor and stator. Thecomponents are usefully introduced in a direction parallel to therotor's axis.

An important performance parameter in rotor-stator homogenizers is thetip speed of the rotor (peripheral velocity). This parameter is afunction both of rotation speed and of rotor diameter. A tip speed of atleast 10 ms⁻¹ is useful, and ideally quicker e.g. ≥20 ms⁻¹, ≥30 ms⁻¹,≥40 ms⁻¹, etc. A tip speed of 40 ms⁻¹ can be readily achieved at 10,000rpm with a small homogenizer or at lower rotation speeds (e.g. 2,000rpm) with a larger homogenizer. Suitable high-shear homogenizers arecommercially available.

For commercial-scale manufacture the homogenizer should ideally have aflow rate of at least 300 L/hr e.g. ≥400 L/hr, ≥500 L/hr, ≥600 L/hr,≥700 L/hr, ≥800 L/hr, ≥900 L/hr, ≥1000 L/hr, ≥2000 L/hr, ≥5000 L/hr, oreven ≥10000 L/hr. Suitable high-capacity homogenizers are commerciallyavailable.

A preferred homogenizer provides a shear rate of between 3×10⁵ and 1×10⁶s⁻¹, e.g. between 3×10⁵ and 7×10⁵ s⁻¹, between 4×10⁵ and 6×10⁵ s⁻¹, e.g.about 5×10⁵ s⁻¹.

Although rotor-stator homogenizers generate relatively little heatduring operation, the homogenizer may be cooled during use. Ideally, thetemperature of the first emulsion is maintained below 60° C. duringhomogenization, e.g. below 45° C.

In some embodiments the first emulsion components may be homogenizedmultiple times (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or moretimes). To avoid the need for a long string of containers andhomogenizers the emulsion components can instead be circulated (e.g.feature (11) in FIG. 1). In particular, the first emulsion may be formedby circulating the first emulsion components through a homogenizer aplurality of times (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100etc times). However, too many cycles may be undesirable as it canproduce re-coalescence as described in reference 14. Thus the size ofoil droplets may be monitored if homogenizer circulation is used tocheck that a desired droplet size is reached and/or that re-coalescenceis not occurring.

Circulation through the homogenizer is advantageous because it canreduce the average size of the oil droplets in the first emulsion.Circulation is also advantageous because it can reduce the number of oildroplets having a size >1.2 μm in the first emulsion. These reductionsin average droplet size and number of droplets >1.2 μm in the firstemulsion can provide advantages in downstream process(es). Inparticular, circulation of the first emulsion components through thehomogenizer can lead to an improved microfluidization process which maythen result in a reduced number of oil droplets having a size >1.2 μm inthe second emulsion, i.e. after microfluidization. This improvement inthe second emulsion parameters can provide improved filtrationperformance. Improved filtration performance may lead to less contentlosses during filtration, e.g. losses of squalene, Tween 80 and Span 85when the oil-in-water emulsion is MF59.

Two particular types of circulation are referred to herein as “type I”and “type II”. Type I circulation is illustrated in FIG. 5, whereas typeII circulation is illustrated in FIG. 6.

The circulation of the first emulsion components may comprise a type Icirculation of transferring the first emulsion components between afirst premix container and a homogenizer. The first premix container maybe from 50 to 500 L in size, e.g. 100 to 400 L, 100 to 300 L, 200 to 300L, 250 L or 280 L. The first premix container may be manufactured fromstainless steel. The type I circulation may be continued for 10 to 60minutes, e.g. 10 to 40 minutes or 20 minutes.

The circulation of the first emulsion components may comprise a type IIcirculation of transferring the first emulsion components from a firstpremix container, through a first homogenizer to a second premixcontainer (optionally having the same properties as the first premixcontainer), and then through a second homogenizer. The secondhomogenizer will usually be the same as the first homogenizer, but insome arrangements the first and second homogenizers are different.Following the pass of the first emulsion components through the secondhomogenizer, the first emulsion components may be transferred back tothe first premix container, for example if the type II circulationprocess is to be repeated. Thus the emulsion components may travel in afigure of eight route between the first and second premix containers viaa single homogenizer (see FIG. 6). Type II circulation may be carriedout a single time or a plurality of times, e.g. 2, 3, 4, 5 etc times.

Type II circulation is advantageous, compared to type I circulation,because it can help to ensure that all of the components of the firstemulsion pass through the homogenizer. Emptying of the first premixcontainer means that the complete emulsion contents have passed throughthe homogenizer, into the second premix container. Similarly, thecontents of the second premix container can be emptied, again ensuringthat they all pass through the homogenizer. Thus the type II arrangementcan conveniently ensure that all of the emulsion components arehomogenized at least twice, which can reduce both the average size ofthe oil droplets and the number of oil droplets having a size >1.2 μm inthe first emulsion. An ideal type II circulation thus involves emptyingthe first premix container and passing substantially all of its contentsthrough the homogenizer into the second premix container, followed byemptying the second premix container and re-passing substantially all ofits contents through the homogenizer back into the first premixcontainer. Thus all particles pass through the homogenizer at leasttwice, whereas this is difficult to achieve with type I circulation.

In some embodiments a combination of type I and type II circulations isused, and this combination can provide a first emulsion with goodcharacteristics. In particular, this combination can greatly reduce ofthe number of oil droplets having a size >1.2 μm in the first emulsion.This combination can comprise any order of type I and II circulation,e.g., type I followed by type II, type II followed by type I, type Ifollowed by type II followed by type I again etc. In one embodiment, thecombination comprises 20 minutes of type I circulation followed by asingle type II circulation, i.e. transferring the circulated firstemulsion components from a first premix container, through a firsthomogenizer to a second premix container, and then through a secondhomogenizer once.

The first and second premix containers may be held under an inert gas,e.g. nitrogen, e.g. at up to 0.5 bar. This can prevent the emulsioncomponents from oxidizing, which is particularly advantageous if one ofthe emulsion components is squalene. This can provide an increase in thestability of the emulsion.

As mentioned above, the initial input for the homogenizer may be anon-homogenized mixture of the first emulsion components. This mixturemay be prepared by mixing the individual first emulsion componentsindividually but, in some embodiments, multiple components can becombined prior to this mixing. For instance, if the emulsion includes asurfactant with a HLB below 10 then this surfactant may be combined withan oil prior to mixing. Similarly, if the emulsion includes a surfactantwith a HLB above 10 then this surfactant may be combined with an aqueouscomponent prior to mixing. Buffer salts may be combined with an aqueouscomponent prior to mixing, or may be added separately.

Methods of the invention may be used at large scale. Thus a method mayinvolve preparing a first emulsion whose volume is greater than 1 litere.g. ≥5 liters, ≥10 liters, ≥20 liters, ≥50 liters, ≥100 liters, ≥250liters, etc.

After its formation, the first emulsion may be microfluidized, or may bestored to await microfluidization.

In some embodiments, in particular those where multiple cycles of steps(i) and (ii) are used, the input for the homogenizer will be the outputof a microfluidizer, such that the first emulsion is microfluidized andthen again subjected to homogenization.

Microfluidization

After its formation the first emulsion is microfluidized in order toreduce its average oil droplet size and/or to reduce the number of oildroplets having a size of >1.2 μm.

Microfluidization instruments reduce average oil droplet size bypropelling streams of input components through geometrically fixedchannels at high pressure and high velocity. The pressure at theentrance to the interaction chamber (also called the “first pressure”)may be substantially constant (i.e. ±15%; e.g. ±10%, ±5%, ±2%) for atleast 85% of the time during which components are fed into themicrofluidizer, e.g. at least 87%, at least 90%, at least 95%, at least99% or 100% of the time during which the emulsion is fed into themicrofluidizer.

In one embodiment, the first pressure is 1300 bar±15% (18 kPSI±15%),i.e. between 1100 bar and 1500 bar (between 15 kPSI and 21 kPSI) for 85%of the time during which the emulsion is fed into the microfluidizer.Two suitable pressure profiles are shown in FIG. 3. In FIG. 3A thepressure is substantially constant for at least 85% of the time, whereasin FIG. 3B the pressure continuously remains substantially constant.

A microfluidization apparatus typically comprises at least oneintensifier pump (preferably two pumps, which may be synchronous) and aninteraction chamber. The intensifier pump, which is ideallyelectric-hydraulic driven, provides high pressure (i.e. the firstpressure) to force an emulsion into and through the interaction chamber.The synchronous nature of the intensifier pumps may be used to providethe substantially constant pressure of the emulsion discussed above,which means that the emulsion droplets are all exposed to substantiallythe same level of shear forces during microfluidization.

One advantage of the use of a substantially constant pressure is that itcan reduce fatigue failures in the microfluidization device, which maylead to longer life of the device. A further advantage of the use of asubstantially constant pressure is that the parameters of the secondemulsion can be improved. In particular, the number of oil dropletshaving a size >1.2 μm present in the second emulsion can be reduced.Furthermore, the average oil droplet size of the second emulsion can bereduced when a substantially constant pressure is used. The reduction inthe average oil droplet size and in the number of oil droplets having asize >1.2 μm in the second emulsion may provide improved filtrationperformance. Improved filtration performance may lead to less contentlosses during filtration, e.g. losses of squalene, Tween 80 and Span 85when the emulsion is MF59.

The interaction chamber may contain a plurality, e.g. 2, 3, 4, 5, 6, 7,8, 9, 10 etc, of fixed geometry channels into which the emulsion passes.The emulsion enters the interaction chamber through an input line whichmay have a diameter of between 200 to 250 μm. The emulsion divides intostreams as it enters the interaction chamber and, under high pressure,accelerates to high velocity. As it passes through the channels, forcesproduced by the high pressure may act to reduce the emulsion's oildroplet size and reduce the number of oil droplets having a size >1.2μm. These forces can include: shear forces, through deformation of theemulsion stream occurring from contact with channel walls; impactforces, through collisions occurring when high velocity emulsion streamscollide with each other; and cavitation forces, through formation andcollapse of cavities within the stream. The interaction chamber usuallyincludes no moving parts. It may include ceramic (e.g. alumina) ordiamond (e.g. polycrystalline diamond) channel surfaces. Other surfacesmay be made of stainless steel.

The fixed geometry of the plurality of channels in the interactionchamber may be “Y” type geometry or “Z” type geometry.

In a Y-type geometry interaction chamber a single input emulsion streamis split into first and second emulsion streams, which are thenrecombined into a single output emulsion stream. Prior to recombination,each of the first and second emulsion streams may independently be splitinto a first and second plurality (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 etc.)of sub-streams. When the emulsion streams are recombined, the first andsecond emulsion streams (or their sub-streams) are ideally flowing insubstantially opposite directions (e.g. the first and second emulsionstreams, or their sub-streams, are flowing in substantially the sameplane (±20°) and the flow direction of the first emulsion stream is180±20° different from the flow direction of the second emulsionstream). The forces produced when the emulsion streams are recombinedmay act to reduce the emulsion's oil droplet size and reduce the numberof oil droplets having a size >1.2 μm.

In a Z-type geometry interaction chamber the emulsion stream passesaround a plurality (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 etc) ofsubstantially right angled corners (i.e. 90±20°). FIG. 4 illustrates aninteraction chamber with Z-type geometry and two right-angled corners inthe direction of flow. During its passage around the corners, an inputemulsion stream may be split into a plurality (e.g. 2, 3, 4, 5, 6, 7, 8,9, 10 etc.) of sub-streams and then recombined into a single outputemulsion stream (e.g. as shown in FIG. 4, with four sub-streams (32)).The split and then recombination (31) may occur at any point betweeninput and output. The forces produced when the emulsion contacts thechannel walls as it passes around the corners may act to reduce theemulsion's oil droplet size and reduce the number of oil droplets havinga size >1.2 μm. An example of a Z-type interaction chamber is the E230Zinteraction chamber from Microfluidics.

In one embodiment, the emulsion stream passes around two substantiallyright angled corners. At the point when the input emulsion stream passesaround the first substantially right angled corner, it is split intofive sub-streams. At the point when the sub-streams pass around thesecond substantially right angled corner, they are recombined into asingle output emulsion stream.

In the prior art it has been usual to use Y-type interaction chambersfor oil-in-water emulsions like those of the present invention. However,we have discovered that it is advantageous to use a Z-type channelgeometry interaction chamber for oil-in-water emulsions because this canlead to a greater reduction in the number of oil droplets having a sizeof >1.2 μm present in the second emulsion compared to a Y-type geometryinteraction chamber. The reduction in number of oil droplets having asize >1.2 μm in the second emulsion can provide improved filtrationperformance. Improved filtration performance may lead to less contentlosses during filtration, e.g. losses of squalene, Tween 80 and Span 85when the emulsion is MF59.

A preferred microfluidization apparatus operates at a pressure between170 bar and 2750 bar (approximately 2500 psi to 40000 psi) e.g. at about345 bar, about 690 bar, about 1380 bar, about 2070 bar, etc.

A preferred microfluidization apparatus operates at a flow rate of up to20 L/min e.g. up to 14 L/min, up to 7 L/min, up to 3.5 L/min, etc.

A preferred microfluidization apparatus has an interaction chamber thatprovides a shear rate in excess of 1×10⁶ s⁻¹ e.g. ≥2.5×10⁶ s⁻¹, ≥5×10⁶s⁻¹, ≥10⁷ s⁻¹ etc.

A microfluidization apparatus can include multiple interaction chambersthat are used in parallel e.g. 2, 3, 4, 5 or more, but it is more usefulto include a single interaction chamber.

The microfluidization device may comprise an auxiliary processing module(APM) comprising at least one channel. The APM contributes to thereduction in the average size of the oil droplets in the emulsion beingpassed through the microfluidization device, although the majority ofthe reduction occurs in the interaction chamber. As mentioned above, theemulsion components are introduced to the interaction chamber by theintensifier pump(s) under a first pressure. The emulsion componentsgenerally exit the APM at a second pressure which is lower than thefirst pressure (e.g. atmospheric pressure). In general, between 80 and95% of the pressure difference between the first and the secondpressures is dropped across the interaction chamber (e.g. from P₁ to P₂in FIG. 4) and 5 to 20% of the pressure difference between the first andthe second pressures is dropped across the auxiliary processing module,e.g. the interaction chamber may provide approximately 90% of thepressure drop while the APM may provide approximately 10% of thepressure drop. If the pressure dropped across the interaction chamberand the pressure dropped across the auxiliary processing module do notaccount for the whole of the pressure difference between the first andthe second pressure, this can be due to a finite pressure drop acrossthe connectors between the interaction chamber and the auxiliaryprocessing module.

The APM usually includes no moving parts. It may include ceramic (e.g.alumina) or diamond (e.g. polycrystalline diamond) channel surfaces.Other surfaces may be made of stainless steel.

The APM is generally positioned downstream of the interaction chamberand may also be positioned sequential to the interaction chamber. In theprior art, APMs are generally positioned downstream of interactionchambers comprising Y-type channels to suppress cavitation and therebyincrease the flowrate in the Y-type chamber by up to 30%. Furthermore,in the prior art APMs are generally positioned upstream of interactionchambers comprising Z-type channels to reduce the size of largeagglomerates. In the latter case, the APM only decreases the flowrate inthe Z-type chambers by up to 3%. However, it has been found thatpositioning the APM downstream of an interaction chamber comprising aplurality of Z-type channels is advantageous in the present inventionbecause it can lead to a greater reduction in average oil droplet sizeand a greater reduction in the number of oil droplets having a sizeof >1.2 μm present in the second emulsion. As discussed above, thereduction in number of oil droplets having a size >1.2 μm in the secondemulsion may provide improved filtration performance. Improvedfiltration performance may lead to less content losses duringfiltration, e.g. losses of squalene, Tween 80 and Span 85 when theoil-in-water emulsion is MF59. A further advantage of this positioningof a Z-type interaction chamber and a downstream APM is that it can leadto a slower pressure decrease after the interaction chamber. The slowerpressure decrease may lead to an increase in product stability becausethere is less gas enclosed in the emulsion.

An APM contains at least one fixed geometry channel into which theemulsion passes. The APM may contain a plurality e.g. 2, 3, 4, 5, 6, 7,8, 9, 10 etc, of fixed geometry channels into which the emulsion passes.The channel or channels of the APM may be linear or non-linear. Suitablenon-linear channels are of “Z” type geometry or “Y” type geometry, whichare the same as those described above for the interaction chamber. Inone embodiment, the channel, or channels, of the APM are of Z-typegeometry. A plurality of Z-type channels divides the emulsion intostreams as it enters the APM.

In contrast to the manufacturer's recommendations, the use of an APMcomprising a plurality of fixed geometry channels is advantageouscompared to a single fixed geometry channel APM because it can lead to agreater reduction in the number of oil droplets having a size >1.2 μmpresent in the second emulsion. As discussed above, the reduction in thenumber of oil droplets having a size >1.2 μm in the second emulsion canprovide improved filtration performance. Improved filtration performancemay lead to less content losses during filtration, e.g. losses ofsqualene, Tween 80 and Span 85 when the oil-in-water emulsion is MF59.

A microfluidization apparatus generates heat during operation, which canraise an emulsion's temperature by 15-20° C. relative to the firstemulsion. Advantageously, therefore, the microfluidized emulsion iscooled as soon as possible. The temperature of the second emulsion maybe maintained below 60° C., e.g. below 45° C. Thus an interactionchamber's output and/or an APM's output may feed into a coolingmechanism, such as a heat exchanger or cooling coil. The distancebetween the output and the cooling mechanism should be kept as short aspossible to shorten the overall time by reducing cooling delays. In oneembodiment, the distance between the output of the microfluidizer andthe cooling mechanism is between 20-30 cm. A cooling mechanism isparticularly useful when an emulsion is subjected to multiplemicrofluidization steps, to prevent over-heating of the emulsion.

The result of microfluidization is an oil-in-water emulsion, the secondemulsion, in which the average size of the oil droplets is 500 nm orless. This average size is particularly useful as it facilitates filtersterilization of the emulsion. Emulsions in which at least 80% by numberof the oil droplets have an average size of 500 nm or less, e.g. 400 nmor less, 300 nm of less, 200 nm or less or 165 nm or less, areparticularly useful. Furthermore, the number of oil droplets in thesecond emulsion having a size >1.2 μm is 5×10¹⁰/ml or less, e.g.5×10⁹/ml or less, 5×10⁸/ml or less or 2×10⁸/ml or less.

The initial input for the microfluidization may be the first emulsion.In some embodiments, however, the microfluidized emulsion is subjectedto microfluidization again, such that multiple rounds ofmicrofluidization occur. In particular, the second emulsion may beformed by circulating the second emulsion components through amicrofluidization device a plurality of times, e.g. 2, 3, 4, 5, 6, 7, 8,9, 10 etc times. The second emulsion may be formed by circulating thesecond emulsion components through a microfluidization device 4 to 7times.

The circulation of the second emulsion components may comprise a type Icirculation of transferring the second emulsion components between afirst emulsion container (optionally having the same properties as thefirst premix container) and the microfluidization device.

The circulation of the second emulsion components may comprise a type IIcirculation of transferring the second emulsion components from a firstemulsion container, through a first microfluidization device to a secondemulsion container (optionally having the same properties as the firstpremix container), and then through a second microfluidization device.

The second microfluidization device may be the same as the firstmicrofluidization device. Alternatively, the second microfluidizationdevice may be different to the first microfluidization device.

The first emulsion container may be the same as the first premixcontainer. Alternatively, the first emulsion container may be the sameas the second premix container.

The second emulsion container may be the same as the first premixcontainer. Alternatively, the second emulsion container may be the sameas the second premix container.

The first emulsion container may be the same as the first premixcontainer and the second emulsion container may be the same as thesecond premix container. Alternatively, the first emulsion container maybe the same as the second premix container and the second emulsioncontainer may be the same as the first premix container.

As an alternative, the first and second emulsion containers may bedifferent to the first and second premix containers.

Following the pass of the second emulsion components through the secondmicrofluidization device, the second emulsion components may betransferred back to the first emulsion container, for example if thetype II circulation process is to be repeated. Type II circulation maybe carried out a single time or a plurality of times, e.g. 2, 3, 4, 5etc times.

Type II circulation is advantageous as it ensures that all the secondemulsion components have passed through the microfluidization device atleast 2 times, which reduces the average size of the oil droplets andthe number of oil droplets having a size >1.2 μm in the second emulsion.

A combination of type I circulation and type II circulation may be usedduring microfluidization. This combination can comprise any order oftype I and II circulation, e.g., type I followed by type II, type IIfollowed by type I, type I followed by type II followed by type I againetc.

The first and second emulsion containers may be held under an inert gas,e.g. up to 0.5 bar of nitrogen. This prevents the emulsion componentsoxidizing, which is particularly advantageous if one of the emulsioncomponents is squalene. This leads to an increase in the stability ofthe emulsion.

Methods of the invention may be used at large scale. Thus a method mayinvolve microfluidizing a volume greater than 1 liter e.g. ≥5 liters,≥10 liters, ≥20 liters, ≥50 liters, ≥100 liters, ≥250 liters, etc.

Filtration

After microfluidization, the second emulsion is filtered. Thisfiltration removes any large oil droplets that have survived thehomogenization and microfluidization procedures. Although small innumber terms, these oil droplets can be large in volume terms and theycan act as nucleation sites for aggregation, leading to emulsiondegradation during storage. Moreover, this filtration step can achievefilter sterilization.

The particular filtration membrane suitable for filter sterilizationdepends on the fluid characteristics of the second emulsion and thedegree of filtration required. A filter's characteristics can affect itssuitability for filtration of the microfluidized emulsion. For example,its pore size and surface characteristics can be important, particularlywhen filtering a squalene-based emulsion.

The pore size of membranes used with the invention should permit passageof the desired droplets while retaining the unwanted droplets. Forexample, it should retain droplets that have a size of ≥1 μm whilepermitting passage of droplets <200 nm. A 0.2 μm or 0.22 μm filter isideal, and can also achieve filter sterilization.

The emulsion may be prefiltered e.g. through a 0.45 μm filter. Theprefiltration and filtration can be achieved in one step by the use ofknown double-layer filters that include a first membrane layer withlarger pores and a second membrane layer with smaller pores.Double-layer filters are particularly useful with the invention. Thefirst layer ideally has a pore size >0.3 μm, such as between 0.3-2 μm orbetween 0.3-1 μm, or between 0.4-0.8 μm, or between 0.5-0.7 μm. A poresize of ≤0.75 μm in the first layer is preferred. Thus the first layermay have a pore size of 0.6 μm or 0.45 μm, for example. The second layerideally has a pore size which is less than 75% of (and ideally less thanhalf of) the first layer's pore size, such as between 25-70% or between25-49% of the first layer's pore size e.g. between 30-45%, such as ⅓ or4/9, of the first layer's pore size. Thus the second layer may have apore size <0.3 μm, such as between 0.15-0.28 μm or between 0.18-0.24 μme.g. a 0.2 μm or 0.22 μm pore size second layer. In one example, thefirst membrane layer with larger pores provides a 0.45 μm filter, whilethe second membrane layer with smaller pores provides a 0.22 μm filter.

The filtration membrane and/or the prefiltration membrane may beasymmetric. An asymmetric membrane is one in which the pore size variesfrom one side of the membrane to the other e.g. in which the pore sizeis larger at the entrance face than at the exit face. One side of theasymmetric membrane may be referred to as the “coarse pored surface”,while the other side of the asymmetric membrane may be referred to asthe “fine pored surface”. In a double-layer filter, one or (ideally)both layers may be asymmetric.

The filtration membrane may be porous or homogeneous. A homogeneousmembrane is usually a dense film ranging from 10 to 200 μm. A porousmembrane has a porous structure. In one embodiment, the filtrationmembrane is porous. In a double-layer filter, both layers may be porous,both layers may be homogenous, or there may be one porous and onehomogenous layer. A preferred double-layer filter is one in which bothlayers are porous.

In one embodiment, the second emulsion is prefiltered through anasymmetric, hydrophilic porous membrane and then filtered throughanother asymmetric hydrophilic porous membrane having smaller pores thanthe prefiltration membrane. This can use a double-layer filter.

The filter membrane(s) may be autoclaved prior to use to ensure that itis sterile.

Filtration membranes are typically made of polymeric support materialssuch as PTFE (poly-tetra-fluoro-ethylene), PES (polyethersulfone), PVP(polyvinyl pyrrolidone), PVDF (polyvinylidene fluoride), nylons(polyamides), PP (polypropylene), celluloses (including celluloseesters), PEEK (polyetheretherketone), nitrocellulose, etc. (although theinvention preferably avoids the use of cellulose-based filters). Thesesupport materials have varying characteristics, with some supports beingintrinsically hydrophobic (e.g. PTFE) and others being intrinsicallyhydrophilic (e.g. cellulose acetates). However, these intrinsiccharacteristics can be modified by treating the membrane surface. Forinstance, it is known to prepare hydrophilized or hydrophobizedmembranes by treating them with other materials (such as other polymers,graphite, silicone, etc.) to coat the membrane surface e.g. see section2.1 of reference 15. In a double-layer filter the two membranes can bemade of different materials or (ideally) of the same material.

An ideal filter for use with the invention has a hydrophilic surface, incontrast to the teaching of references 9-12 that hydrophobic(polysulfone) filters should be used. Filters with hydrophilic surfacescan be formed from hydrophilic materials, or by hydrophilization ofhydrophobic materials, and a preferred filter for use with the inventionis a hydrophilic polyethersulfone membrane. Several different methodsare known to transform hydrophobic PES membranes into hydrophilic PESmembranes. Often it is achieved by coating the membrane with ahydrophilic polymer. To provide permanent attachment of the hydrophilicpolymer to the PES a hydrophilic coating layer is usually subjectedeither to a cross-linking reaction or to grafting. Reference 15discloses a process for modifying the surface properties of ahydrophobic polymer having functionalizable chain ends, comprisingcontacting the polymer with a solution of a linker moiety to form acovalent link, and then contacting the reacted hydrophobic polymer witha solution of a modifying agent. Reference 16 discloses a method of PESmembrane hydrophilization by direct membrane coating, involvingpre-wetting with alcohol, and then soaking in an aqueous solutioncontaining a hydrophilic monomer, a polyfunctional monomer(cross-linker) and a polymerization initiator. The monomer andcross-linker are then polymerized using thermal- or UV-initiatedpolymerization to form a coating of cross-linked hydrophilic polymer onthe membrane surface. Similarly, references 17 and 18 disclose coating aPES membrane by soaking it in an aqueous solution of hydrophilic polymer(polyalkylene oxide) and at least one polyfunctional monomer(cross-linker), and then polymerizing a monomer to provide anon-extractable hydrophilic coating. Reference 19 describes thehydrophilization of PES membrane by a grafting reaction in which a PESmembrane is submitted to low-temperature helium plasma treatmentfollowed by grafting of hydrophilic monomer N-vinyl-2-pyrrolidone (NVP)onto the membrane surface. Further such processes are disclosed inreferences 20 to 26.

In methods that do not rely on coating, PES can be dissolved in asolvent, blended with a soluble hydrophilic additive, and then theblended solution is used for casting a hydrophilic membrane e.g. byprecipitation or by initiating co-polymerization. Such methods aredisclosed in references 27 to 33. For example, reference 33 discloses amethod of preparing a hydrophilic charge-modified membrane that has lowmembrane extractables and allows fast recovery of ultrapure waterresistivity, having a cross-linked inter-penetrating polymer networkstructure formed making a polymer solution of a blend of PES, PVP,polyethyleneimine, and aliphatic diglycidyl ether, forming a thin filmof the solution, and precipitating the film as a membrane. A similarprocess is disclosed in reference 34.

Hybrid approaches can be used, in which hydrophilic additives arepresent during membrane formation and are also added later as a coatinge.g. see reference 35.

Hydrophilization of PES membrane can also be achieved by treatment withlow temperature plasmas. Reference 36 describes hydrophilic modificationof PES membrane by treatment with low temperature CO₂-plasma.

Hydrophilization of PES membrane can also be achieved by oxidation, asdescribed in reference 37. This method involves pre-wetting ahydrophobic PES membrane in a liquid having a low surface tension,exposing the wet PES membrane to an aqueous solution of oxidizer, andthen heating.

Phase inversion can also be used, as described in reference 38.

An ideal hydrophilic PES membrane can be obtained by treatment of PES(hydrophobic) with PVP (hydrophilic). Treatment with PEG (hydrophilic)instead of PVP has been found to give a hydrophilized PES membrane thatis easily fouled (particularly when using a squalene-containingemulsion) and also disadvantageously releases formaldehyde duringautoclaving.

A preferred double-layer filter has a first hydrophilic PES membrane anda second hydrophilic PES membrane. Embodiments are not preferred (andmight sometimes be disclaimed) in which filtration is first via adouble-layer cellulose acetate filter and then through a seconddouble-layer cellulose acetate filter, where the smaller pore size ofthe first filter is larger than the larger pore size of the secondfilter, and where the second double-layer filter achieves filtersterilization.

Known hydrophilic membranes include Bioassure (from Cuno); EverLUX™polyethersulfone; STyLUX™ polyethersulfone (both from Meissner); MillexGV, Millex HP, Millipak 60, Millipak 200 and Durapore CVGL01TP3membranes (from Millipore); Fluorodyne™ EX EDF Membrane, Supor™ EAV;Supor™ EBV, Supor™ EKV (all from Pall); Sartopore™ (from Sartorius);Sterlitech's hydrophilic PES membrane; and Wolftechnik's WFPES PESmembrane.

During filtration, the emulsion may be maintained at a temperature of40° C. or less, e.g. 30° C. or less, to facilitate successful sterilefiltration. Some emulsions may not pass through a sterile filter whenthey are at a temperature of greater than 40° C.

It is advantageous to carry out the filtration step within 24 hours,e.g. within 18 hours, within 12 hours, within 6 hours, within 2 hours,within 30 minutes, of producing the second emulsion because after thistime it may not be possible to pass the second emulsion through thesterile filter without clogging the filter, as discussed in reference39.

Methods of the invention may be used at large scale. Thus a method mayinvolve filtering a volume greater than 1 liter e.g. ≥5 liters, ≥10liters, ≥20 liters, ≥50 liters, ≥100 liters, ≥250 liters, etc.

The Final Emulsion

The result of microfluidization and filtration is an oil-in-wateremulsion in which the average size of the oil droplets may be less than220 nm, e.g. 155±20 nm, 155±10 nm or 155±5 nm, and in which the numberof oil droplets having a size >1.2 μm may be 5×10⁸/ml or less, e.g.5×10⁷/ml or less, 5×10⁶/ml or less, 2×10⁶/ml or less or 5×10⁵/ml orless.

The average oil droplet size of emulsions described herein (includingthe first and second emulsions) is generally not less than 50 nm.

Methods of the invention may be used at large scale. Thus a method mayinvolve preparing a final emulsion with a volume greater than 1 litere.g. ≥5 liters, ≥10 liters, ≥20 liters, ≥50 liters, ≥100 liters, ≥250liters, etc.

Once the oil-in-water emulsion has been formed, it may be transferredinto sterile glass bottles. The glass bottles may be 5 L, 8 L, or 10 Lin size. Alternatively, the oil-in-water may be transferred into asterile flexible bag (flex bag). The flex bag may be 50 L, 100 L or 250L in size. In addition, the flex bag may be fitted with one or moresterile connectors to connect the flex bag to the system. The use of aflex bag with a sterile connectors is advantageous compared to glassbottles because the flex bag is larger then the glass bottles meaningthat it may not be necessary to change the flex bag to store all theemulsion manufactured in a single batch. This can provide a sterileclosed system for the manufacture of the emulsion which may reduce thechance of impurities being present in the final emulsion. This can beparticularly important if the final emulsion is used for pharmaceuticalpurposes, e.g. if the final emulsion is the MF59 adjuvant.

Preferred amounts of oil (% by volume) in the final emulsion are between2-20% e.g. about 10%. A squalene content of about 5% or about 10% isparticularly useful. A squalene content (w/v) of between 30-50 mg/ml isuseful e.g. between 35-45 mg/ml, 36-42 mg/ml, 38-40 mg/ml, etc.

Preferred amounts of surfactants (% by weight) in the final emulsionare: polyoxyethylene sorbitan esters (such as Tween 80) 0.02 to 2%, inparticular about 0.5% or about 1%; sorbitan esters (such as Span 85)0.02 to 2%, in particular about 0.5% or about 1%; octyl- or nonylphenoxypolyoxyethanols (such as Triton X-100) 0.001 to 0.1%, in particular0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%,preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%. Apolysorbate 80 content (w/v) of between 4-6 mg/ml is useful e.g. between4.1-5.3 mg/ml. A sorbitan trioleate content (w/v) of between 4-6 mg/mlis useful e.g. between 4.1-5.3 mg/ml.

The process is particularly useful for preparing any of the followingoil-in-water emulsions:

-   -   An emulsion comprising squalene, polysorbate 80 (Tween 80), and        sorbitan trioleate (Span 85). The composition of the emulsion by        volume can be about 5% squalene, about 0.5% polysorbate 80 and        about 0.5% sorbitan trioleate. In weight terms, these amounts        become 4.3% squalene, 0.5% polysorbate 80 and 0.48% sorbitan        trioleate. This adjuvant is known as ‘MF59’. The MF59 emulsion        advantageously includes citrate ions e.g. 10 mM sodium citrate        buffer.    -   Emulsions comprising squalene, an α-tocopherol (ideally        DL-α-tocopherol), and polysorbate 80. These emulsions may have        (by weight) from 2 to 10% squalene, from 2 to 10% α-tocopherol        and from 0.3 to 3% polysorbate 80 e.g. 4.3% squalene, 4.7%        α-tocopherol, 1.9% polysorbate 80. The weight ratio of        squalene:tocopherol is preferably ≤1 (e.g. 0.90) as this        provides a more stable emulsion. Squalene and polysorbate 80 may        be present volume ratio of about 5:2, or at a weight ratio of        about 11:5. One such emulsion can be made by dissolving        polysorbate 80 in PBS to give a 2% solution, then mixing 90 ml        of this solution with a mixture of (5 g of DL-α-tocopherol and 5        ml squalene), then microfluidizing the mixture. The resulting        emulsion may have submicron oil droplets e.g. with a size        between 100 and 250 nm, preferably about 180 nm.    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a        3-O-deacylated monophosphoryl lipid A (‘3d-MPL’), although        3d-MPL-containing emulsions are not preferred in the present        invention (and might sometimes be disclaimed). The emulsion may        contain a phosphate buffer.    -   An emulsion comprising squalene, a polysorbate (e.g. polysorbate        80), a Triton detergent (e.g. Triton X-100) and a tocopherol        (e.g. an α-tocopherol succinate). The emulsion may include these        three components at a mass ratio of about 75:11:10 (e.g. 750        μg/ml polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include a 3d-MPL. The emulsion may also include a        saponin, such as QS21. The aqueous phase may contain a phosphate        buffer.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [40]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. It may also        include a TLR4 agonist, such as one whose chemical structure        does not include a sugar ring [41]. Such emulsions may be        lyophilized.

The compositions of these emulsions, expressed above in percentageterms, may be modified by dilution or concentration (e.g. by an integer,such as 2 or 3 or by a fraction, such as ⅔ or ¾), in which their ratiosstay the same. For instance, a 2-fold concentrated MF59 would have about10% squalene, about 1% polysorbate 80 and about 1% sorbitan trioleate.Concentrated forms can be diluted (e.g. with an antigen solution) togive a desired final concentration of emulsion.

Emulsions of the invention are ideally stored at between 2° C. and 8° C.They should not be frozen. They should ideally be kept out of directlight. In particular, squalene-containing emulsions and vaccines of theinvention should be protected to avoid photochemical breakdown ofsqualene. If emulsions of the invention are stored then this ispreferably in an inert atmosphere e.g. N₂ or argon.

Vaccines

Although it is possible to administer oil-in-water emulsion adjuvants ontheir own to patients (e.g. to provide an adjuvant effect for an antigenthat has been separately administered to the patient), it is more usualto admix the adjuvant with an antigen prior to administration, to forman immunogenic composition e.g. a vaccine. Mixing of emulsion andantigen may take place extemporaneously, at the time of use, or can takeplace during vaccine manufacture, prior to filling. The methods of theinvention can be applied in both situations.

Thus a method of the invention may include a further process step ofadmixing the emulsion with an antigen component. As an alternative, itmay include a further step of packaging the adjuvant into a kit as a kitcomponent together with an antigen component.

Overall, therefore, the invention can be used when preparing mixedvaccines or when preparing kits including antigen and adjuvant ready formixing. Where mixing takes place during manufacture then the volumes ofbulk antigen and emulsion that are mixed will typically be greater than1 liter e.g. ≥5 liters, ≥10 liters, ≥20 liters, ≥50 liters, ≥100 liters,≥250 liters, etc. Where mixing takes place at the point of use then thevolumes that are mixed will typically be smaller than 1 milliliter e.g.≤0.6 ml, ≤0.5 ml, ≤0.4 ml, ≤0.3 ml, ≤0.2 ml, etc. In both cases it isusual for substantially equal volumes of emulsion and antigen solutionto be mixed i.e. substantially 1:1 (e.g. between 1.1:1 and 1:1.1,preferably between 1.05:1 and 1:1.05, and more preferably between1.025:1 and 1:1.025). In some embodiments, however, an excess ofemulsion or an excess of antigen may be used [42]. Where an excessvolume of one component is used, the excess will generally be at least1.5:1 e.g. ≥2:1, ≥2.5:1, ≥3:1, ≥4:1, ≥5:1, etc.

Where antigen and adjuvant are presented as separate components within akit, they are physically separate from each other within the kit, andthis separation can be achieved in various ways. For instance, thecomponents may be in separate containers, such as vials. The contents oftwo vials can then be mixed when needed e.g. by removing the contents ofone vial and adding them to the other vial, or by separately removingthe contents of both vials and mixing them in a third container.

In another arrangement, one of the kit components is in a syringe andthe other is in a container such as a vial. The syringe can be used(e.g. with a needle) to insert its contents into the vial for mixing,and the mixture can then be withdrawn into the syringe. The mixedcontents of the syringe can then be administered to a patient, typicallythrough a new sterile needle. Packing one component in a syringeeliminates the need for using a separate syringe for patientadministration.

In another preferred arrangement, the two kit components are heldtogether but separately in the same syringe e.g. a dual-chamber syringe,such as those disclosed in references 43-50 etc. When the syringe isactuated (e.g. during administration to a patient) then the contents ofthe two chambers are mixed. This arrangement avoids the need for aseparate mixing step at time of use.

The contents of the various kit components will generally all be inliquid form. In some arrangements, a component (typically the antigencomponent rather than the emulsion component) is in dry form (e.g. in alyophilized form), with the other component being in liquid form. Thetwo components can be mixed in order to reactivate the dry component andgive a liquid composition for administration to a patient. A lyophilizedcomponent will typically be located within a vial rather than a syringe.Dried components may include stabilizers such as lactose, sucrose ormannitol, as well as mixtures thereof e.g. lactose/sucrose mixtures,sucrose/mannitol mixtures, etc. One possible arrangement uses a liquidemulsion component in a pre-filled syringe and a lyophilized antigencomponent in a vial.

If vaccines contain components in addition to emulsion and antigen thenthese further components may be included in one these two kitcomponents, or may be part of a third kit component.

Suitable containers for mixed vaccines of the invention, or forindividual kit components, include vials and disposable syringes. Thesecontainers should be sterile.

Where a composition/component is located in a vial, the vial ispreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex-sensitive patients, vials are preferably sealed with a latex-freestopper, and the absence of latex in all packaging material ispreferred. In one embodiment, a vial has a butyl rubber stopper. Thevial may include a single dose of vaccine/component, or it may includemore than one dose (a ‘multidose’ vial) e.g. 10 doses. In oneembodiment, a vial includes 10×0.25 ml doses of emulsion. Preferredvials are made of colorless glass.

A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial (e.g. to reconstitute lyophilized materialtherein), and the contents of the vial can be removed back into thesyringe. After removal of the syringe from the vial, a needle can thenbe attached and the composition can be administered to a patient. Thecap is preferably located inside a seal or cover, such that the seal orcover has to be removed before the cap can be accessed.

Where a composition/component is packaged into a syringe, the syringewill not normally have a needle attached to it, although a separateneedle may be supplied with the syringe for assembly and use. Safetyneedles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and ⅝-inch25-gauge needles are typical. Syringes may be provided with peel-offlabels on which the lot number, influenza season and expiration date ofthe contents may be printed, to facilitate record keeping. The plungerin the syringe preferably has a stopper to prevent the plunger frombeing accidentally removed during aspiration. The syringes may have alatex rubber cap and/or plunger. Disposable syringes contain a singledose of vaccine. The syringe will generally have a tip cap to seal thetip prior to attachment of a needle, and the tip cap is preferably madeof a butyl rubber. If the syringe and needle are packaged separatelythen the needle is preferably fitted with a butyl rubber shield.

The emulsion may be diluted with a buffer prior to packaging into a vialor a syringe. Typical buffers include: a phosphate buffer; a Trisbuffer; a borate buffer; a succinate buffer; a histidine buffer; or acitrate buffer. Dilution can reduce the concentration of the adjuvant'scomponents while retaining their relative proportions e.g. to provide a“half-strength” adjuvant.

Containers may be marked to show a half-dose volume e.g. to facilitatedelivery to children. For instance, a syringe containing a 0.5 ml dosemay have a mark showing a 0.25 ml volume.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

Various antigens can be used with oil-in-water emulsions, including butnot limited to: viral antigens, such as viral surface proteins;bacterial antigens, such as protein and/or saccharide antigens; fungalantigens; parasite antigens; and tumor antigens. The invention isparticularly useful for vaccines against influenza virus, HIV, hookworm,hepatitis B virus, herpes simplex virus, rabies, respiratory syncytialvirus, cytomegalovirus, Staphylococcus aureus, chlamydia, SARScoronavirus, varicella zoster virus, Streptococcus pneumoniae, Neisseriameningitidis, Mycobacterium tuberculosis, Bacillus anthracis, EpsteinBarr virus, human papillomavirus, etc. For example:

-   -   Influenza virus antigens. These may take the form of a live        virus or an inactivated virus. Where an inactivated virus is        used, the vaccine may comprise whole virion, split virion, or        purified surface antigens (including hemagglutinin and, usually,        also including neuraminidase). Influenza antigens can also be        presented in the form of virosomes. The antigens may have any        hemagglutinin subtype, selected from H1, H2, H3, H4, H5, H6, H7,        H8, H9, H10, H11, H12, H13, H14, H15 and/or H16. Vaccine may        include antigen(s) from one or more (e.g. 1, 2, 3, 4 or more)        influenza virus strains, including influenza A virus and/or        influenza B virus, e.g. a monovalent A/H5N1 or A/H1N1 vaccine,        or a trivalent A/H1N1+A/H3N2+B vaccine. The influenza virus may        be a reassortant strain, and may have been obtained by reverse        genetics techniques [e.g. 51-55]. Thus the virus may include one        or more RNA segments from a A/PR/8/34 virus (typically 6        segments from A/PR/8/34, with the HA and N segments being from a        vaccine strain, i.e. a 6:2 reassortant). The viruses used as the        source of the antigens can be grown either on eggs (e.g.        embryonated hen eggs) or on cell culture. Where cell culture is        used, the cell substrate will typically be a mammalian cell        line, such as MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6; WI-38;        etc. Preferred mammalian cell lines for growing influenza        viruses include: MDCK cells [56-59], derived from Madin Darby        canine kidney; Vero cells [60-62], derived from African green        monkey kidney; or PER.C6 cells [63], derived from human        embryonic retinoblasts. Where virus has been grown on a        mammalian cell line then the composition will advantageously be        free from egg proteins (e.g. ovalbumin and ovomucoid) and from        chicken DNA, thereby reducing allergenicity. Unit doses of        vaccine are typically standardized by reference to hemagglutinin        (HA) content, typically measured by SRID. Existing vaccines        typically contain about 15 μg of HA per strain, although lower        doses can be used, particularly when using an adjuvant.        Fractional doses such as ½ (i.e. 7.5 μg HA per strain), ¼ and ⅛        have been used [64, 65], as have higher doses (e.g. 3× or 9×        doses [66, 67]). Thus vaccines may include between 0.1 and 150        μg of HA per influenza strain, preferably between 0.1 and 50 μg        e.g. 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc.        Particular doses include e.g. about 15, about 10, about 7.5,        about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc. per        strain.    -   Human immunodeficiency virus, including HIV-1 and HIV-2. The        antigen will typically be an envelope antigen.    -   Hepatitis B virus surface antigens. This antigen is preferably        obtained by recombinant DNA methods e.g. after expression in a        Saccharomyces cerevisiae yeast. Unlike native viral HBsAg, the        recombinant yeast-expressed antigen is non-glycosylated. It can        be in the form of substantially-spherical particles (average        diameter of about 20 nm), including a lipid matrix comprising        phospholipids. Unlike native HBsAg particles, the        yeast-expressed particles may include phosphatidylinositol. The        HBsAg may be from any of subtypes ayw1, ayw2, ayw3, ayw4, ayr,        adw2, adw4, adrq− and adrq+.    -   Hookworm, particularly as seen in canines (Ancylostoma caninum).        This antigen may be recombinant Ac-MTP-1 (astacin-like        metalloprotease) and/or an aspartic hemoglobinase (Ac-APR-1),        which may be expressed in a baculovirus/insect cell system as a        secreted protein [68, 69].    -   Herpes simplex virus antigens (HSV). A preferred HSV antigen for        use with the invention is membrane glycoprotein gD. It is        preferred to use gD from a HSV-2 strain (‘gD2’ antigen). The        composition can use a form of gD in which the C-terminal        membrane anchor region has been deleted [70] e.g. a truncated gD        comprising amino acids 1-306 of the natural protein with the        addition of aparagine and glutamine at the C-terminus. This form        of the protein includes the signal peptide which is cleaved to        yield a mature 283 amino acid protein. Deletion of the anchor        allows the protein to be prepared in soluble form.    -   Human papillomavirus antigens (HPV). Preferred HPV antigens for        use with the invention are L1 capsid proteins, which can        assemble to form structures known as virus-like particles        (VLPs). The VLPs can be produced by recombinant expression of L1        in yeast cells (e.g. in S. cerevisiae) or in insect cells (e.g.        in Spodoptera cells, such as S. frugiperda, or in Drosophila        cells). For yeast cells, plasmid vectors can carry the L1        gene(s); for insect cells, baculovirus vectors can carry the L1        gene(s). More preferably, the composition includes L1 VLPs from        both HPV-16 and HPV-18 strains. This bivalent combination has        been shown to be highly effective [71]. In addition to HPV-16        and HPV-18 strains, it is also possible to include L1 VLPs from        HPV-6 and HPV-11 strains. The use of oncogenic HPV strains is        also possible. A vaccine may include between 20-60 μg/ml (e.g.        about 40 μg/ml) of L1 per HPV strain.    -   Anthrax antigens. Anthrax is caused by Bacillus anthraces.        Suitable B. anthracis antigens include A-components (lethal        factor (LF) and edema factor (EF)), both of which can share a        common B-component known as protective antigen (PA). The        antigens may optionally be detoxified. Further details can be        found in references [72 to 74].    -   S. aureus antigens. A variety of S. aureus antigens are known.        Suitable antigens include capsular saccharides (e.g. from a type        5 and/or type 8 strain) and proteins (e.g. IsdB, Hla, etc.).        Capsular saccharide antigens are ideally conjugated to a carrier        protein.    -   S. pneumoniae antigens. A variety of S. pneumoniae antigens are        known. Suitable antigens include capsular saccharides (e.g. from        one or more of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F,        and/or 23F) and proteins (e.g. pneumolysin, detoxified        pneumolysin, polyhistidine triad protein D (PhtD), etc.).        Capsular saccharide antigens are ideally conjugated to a carrier        protein.    -   Cancer antigens. A variety of tumour-specific antigens are        known. The invention may be used with antigens that elicit an        immunotherapeutic response against lung cancer, melanoma, breast        cancer, prostate cancer, etc.

A solution of the antigen will normally be mixed with the emulsion e.g.at a 1:1 volume ratio. This mixing can either be performed by a vaccinemanufacturer, prior to filling, or can be performed at the point of use,by a healthcare worker.

Pharmaceutical Compositions

Compositions made using the methods of the invention arepharmaceutically acceptable. They may include components in addition tothe emulsion and the optional antigen.

The composition may include a preservative such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (i.e. less than 5 μg/ml) mercurial material e.g.thiomersal-free [75, 76]. Vaccines and components containing no mercuryare more preferred.

The pH of a composition will generally be between 5.0 and 8.1, and moretypically between 6.0 and 8.0 e.g. between 6.5 and 7.5. A process of theinvention may therefore include a step of adjusting the pH of thevaccine prior to packaging.

The composition is preferably sterile. The composition is preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose. The composition is preferablygluten free.

The composition may include material for a single immunization, or mayinclude material for multiple immunizations (i.e. a ‘multidose’ kit).The inclusion of a preservative is preferred in multidose arrangements.

Vaccines are typically administered in a dosage volume of about 0.5 ml,although a half dose (i.e. about 0.25 ml) may be administered tochildren.

Methods of Treatment, and Administration of the Vaccine

The invention provides kits and compositions prepared using the methodsof the invention. The compositions prepared according to the methods ofthe invention are suitable for administration to human patients, and theinvention provides a method of raising an immune response in a patient,comprising the step of administering such a composition to the patient.

The invention also provides these kits and compositions for use asmedicaments.

The invention also provides the use of: (i) an aqueous preparation of anantigen; and (ii) an oil-in-water emulsion prepared according to theinvention, in the manufacture of a medicament for raising an immuneresponse in a patient.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.

The compositions can be administered in various ways. The most preferredimmunization route is by intramuscular injection (e.g. into the arm orleg), but other available routes include subcutaneous injection,intranasal [77-79], oral [80], intradermal [81, 82], transcutaneous,transdermal [83], etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. The patient may be less than 1 year old, 1-5 yearsold, 5-15 years old, 15-55 years old, or at least 55 years old. Thepatient may be elderly (e.g. ≥50 years old, preferably ≥65 years), theyoung (e.g. ≤5 years old), hospitalized patients, healthcare workers,armed service and military personnel, pregnant women, the chronicallyill, immunodeficient patients, and people travelling abroad. Thevaccines are not suitable solely for these groups, however, and may beused more generally in a population.

Vaccines of the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) other vaccines.

Intermediate Processes

The invention also provides a method for the manufacture of anoil-in-water emulsion, comprising microfluidization of a first emulsionto form a second emulsion and then filtration of the second emulsion.The first emulsion has the characteristics described above.

The invention also provides a method for the manufacture of anoil-in-water emulsion, comprising filtration of a second emulsion, i.e.a microfluidized emulsion. The microfluidised emulsion has thecharacteristics described above.

The invention also provides a method for the manufacture of a vaccine,comprising combining an emulsion with an antigen, where the emulsion hasthe characteristics described above.

SPECIFIC EMBODIMENTS

Specific embodiments of the present invention include:

-   -   A method for the manufacture of a oil-in-water emulsion        comprising squalene, comprising steps of (i) formation of a        first emulsion having a first average oil droplet size; (ii)        microfluidization of the first emulsion to form a second        emulsion having a second average oil droplet size which is less        than the first average oil droplet size; and (iii) filtration of        the second emulsion using a hydrophilic membrane.    -   A method for the manufacture of a oil-in-water emulsion,        comprising steps of (i) formation of a first emulsion having a        first average oil droplet size of 5000 nm or less; (ii)        microfluidization of the first emulsion to form a second        emulsion having a second average oil droplet size which is less        than the first average oil droplet size; and (iii) filtration of        the second emulsion using a hydrophilic membrane.    -   A method for the manufacture of a oil-in-water emulsion,        comprising steps of (i) formation of a first emulsion having a        first average oil droplet size; (ii) microfluidization of the        first emulsion to form a second emulsion having a second average        oil droplet size which is less than the first average oil        droplet size; and (iii) filtration of the second emulsion using        a hydrophilic polyethersulfone membrane.    -   A method for the manufacture of an oil-in-water emulsion        comprising squalene, the method comprising the step of (i)        formation of a first emulsion having a first average oil droplet        size using a homogenizer, wherein the first emulsion is formed        by circulating the first emulsion components through a        homogenizer a plurality of times.    -   A method for the manufacture of an oil-in-water emulsion        comprising squalene, the method comprising the step of (b)        microfluidization of a first emulsion having a first average oil        droplet size to form a second emulsion having a second average        oil droplet size which is less than the first average oil        droplet size, wherein the second emulsion is formed by        circulating the second emulsion components by transferring the        second emulsion components from a first emulsion container,        through a first microfluidization device to a second emulsion        container, and then through a second microfluidization device,        wherein the first and second microfluidization devices are the        same.    -   A method for the manufacture of an oil-in-water emulsion        comprising: passing a first emulsion having a first average oil        droplet size through a microfluidization device to form a second        emulsion having a second average oil droplet size which is less        than the first average oil droplet size; wherein the        microfluidization device comprises an interaction chamber which        comprises a plurality of Z-type channels and an auxiliary        processing module comprising at least one channel; wherein the        auxiliary processing module is positioned downstream of the        interaction chamber.    -   A method for the manufacture of an oil-in-water emulsion        comprising the step of passing a first emulsion having a first        average oil droplet size through a microfluidization device to        form a second emulsion having a second average oil droplet size        which is less than the first average oil droplet size; wherein        the microfluidization device comprises an interaction chamber        and an auxiliary processing module comprising a plurality of        channels.    -   A method for the manufacture of an oil-in-water emulsion        comprising the step of passing a first emulsion having a first        average oil droplet size through a microfluidization device to        form a second emulsion having a second average oil droplet size        which is less than the first average oil droplet size, wherein        the microfluidization device comprises an interaction chamber        and wherein the pressure of the emulsion components at the        entrance to the interaction chamber is substantially constant        for at least 85% of the time during which the emulsion is fed        into the microfluidizer.    -   A method for the manufacture of a oil-in-water emulsion,        comprising the step of formation of a first emulsion having a        first average oil droplet size, wherein formation of the first        emulsion is carried out under an inert gas, e.g. nitrogen, e.g.        at a pressure of up to 0.5 bar.    -   A method for the manufacture of a oil-in-water emulsion,        comprising the step of passing a first emulsion having a first        average oil droplet size through a microfluidization device to        form a second emulsion having a second average oil droplet size        which is less than the first average oil droplet size, wherein        formation of the second emulsion is carried out under an inert        gas, e.g. nitrogen, e.g. at a pressure of up to 0.5 bar.    -   A method for the manufacture of a oil-in-water emulsion,        comprising steps of (i) formation of a first emulsion having a        first average oil droplet size; (ii) microfluidization of the        first emulsion to form a second emulsion having a second average        oil droplet size which is less than the first average oil        droplet size; (iii) filtration of the second emulsion; (iv)        transfer of the oil-in-water emulsion into a sterile flex bag.        General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, χ±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encaphalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a specific example of a homogenizer that can be used toform a first emulsion.

FIG. 2 shows detail of a rotor and stator that can be used in such ahomogenizer.

FIG. 3 shows two pressure profiles for a synchronous intensifier pumpmode.

FIG. 4 shows a Z-type channel interaction chamber.

FIG. 5 shows a type I circulation, whereas FIG. 6 shows a type IIcirculation. Containers are labeled as “C” whereas a homogenizer islabeled as “H”. Direction and order of fluid movements are shown.

In FIG. 6 the homogenizer has two input arrows and two output arrows butin reality the homogenizer has a single input channel and a singleoutput channel.

MODES FOR CARRYING OUT THE INVENTION Example 1

A microfluidized emulsion comprising squalene, polysorbate 80, sorbitantrioleate and sodium citrate buffer was prepared according to thepresent invention. The emulsion was microfluidized until it had anaverage oil droplet size of 165 nm or less and a number of oil dropletshaving a size >1.2 μm of 5×10⁸/ml or less.

The emulsion was filtered through a sterilizing grade filter cartridge(filter A) having a prefilter membrane of hydrophilic asymmetric porouspolyethersulfone having a pore size of 0.45 μm and an endfilter membraneof hydrophilic asymmetric porous polyethersulfone having a pore size of0.2 μm. During filtration, the emulsion was maintained at a temperatureof 40±5° C.

The above process was carried out for four separate runs and thecharacteristics of the filtered emulsions were measured and are shown inTable 1.

TABLE 1 Actual Value Test parameter Run 1 Run 2 Run 3 Run 4 Average oildroplet size 148 144 144 150 Number of oil droplets 0.08 × 10⁶ 0.08 ×10⁶ 0.12 × 10⁶ 0.20 × 10⁶ having a size > 1.2 μm

As shown in Table 1, filter A consistently reduced the average size ofthe oil droplets in the emulsion. Furthermore, filter A consistentlyreduced the number of oil droplets having a size >1.2 μm in the emulsionby approximately 10³ fold.

Example 2

The same microfluidized emulsion as used for example 1 was filteredthrough a different sterilizing grade filter cartridge (filter B).Filter B had a prefilter membrane of hydrophilic asymmetric porouspolyethersulfone and an endfilter membrane of hydrophilic asymmetricporous polyethersulfone having a pore size of 0.2 μm. During filtration,the emulsion was maintained at a temperature of 40±5° C. This processwas carried out for four separate runs and the characteristics of thefiltered emulsions were measured and are shown in Table 2.

TABLE 2 Measured value Test parameter Run 1 Run 2 Run 3 Run 4 Averageoil droplet size 142 143 141 141 Number of oil droplets 0.23 × 10⁶ 0.15× 10⁶ 0.20 × 10⁶ 0.23 × 10⁶ having a size > 1.2 μm

As shown in Table 2, filter B consistently reduced the average size ofthe oil droplets in the emulsion. Furthermore, filter B consistentlyreduced the number of oil droplets having a size >1.2 μm in the emulsionby approximately 10³ fold.

From examples 1 and 2, it can be seen that filter B results in aslightly lower oil droplet size but a slightly greater number of oildroplets having a size greater than 1.2 μm. However, both filters A andB showed excellent results.

Example 3

The same microfluidized emulsion as used for example 1 was filteredthrough another different sterilizing grade filter cartridge (filter C).Filter C had a prefilter membrane of hydrophilic asymmetric porouspolyethersulfone having a pore size of 0.45 μm and an endfilter membraneof hydrophilic asymmetric porous polyethersulfone having a pore size of0.2 μm. During filtration, the emulsion was maintained at a temperatureof 40±5° C.

In addition, the same microfluidized emulsion as used for example 1 wasfiltered through another different sterilizing grade filter cartridge(filter D). Filter D had a prefilter membrane of hydrophilic asymmetricporous polyethersulfone and an endfilter membrane of hydrophilic porousPVDF. During filtration, the emulsion was maintained at a temperature of40±5° C.

Filter C showed excellent filtration results providing a filteredemulsion having an average oil droplet size of 155±20 nm and a number ofoil droplets having a size >1.2 μm of 5×10⁶/ml or less. Although, filterD also provided a filtered emulsion meeting the above criteria, it wasfound to block more quickly, thus necessitating the replacement of thefilter D membrane. Thus, all polyethersulfone filters were superior tothis PVDF filter.

Example 4

Ten different hydrophilic membranes, from various manufacturers, weretested for filtering a microfluidized emulsion comprising squalene,polysorbate 80, sorbitan trioleate and sodium citrate buffer. Thefilters were numbered 1 to 10 as shown in Table 3 (NB: filter 1 is thesame as filter C in Example 3 above; filter 2 is filter D; filter 9 isfilter A; and filter 10 is filter B).

The yield of emulsion after filtration of 50 liters of emulsion wasmeasured to determine if the filters were suitable for industrial-scaleuse. The % of input emulsion which was recovered after filtration was asfollows:

TABLE 3 1 2 3 4 5 6 7 8 9 10 % 50 16 8 8 8 13 4 82 88 89

A low recovery % indicates that the filter retains the emulsion, forinstance because of blocking. It is clear that only filters 1, 8, 9 and10 (i.e. filters A, B & C from above, plus one further filter which issimilar to filter A but has a larger pore size in the first layer) gaveyields of ≥50%, and that the yields that are most practicable for usingat an industrial scale are filters 8-10. Filters 1, 8, 9 and 10 are alldouble-layer hydrophilic PES membranes, prepared by three differentmanufacturers. The first layers in these four membranes are either 0.45μm or 0.6 μm and the second layer is 0.2 μm. The best results were seenwhen at least one of the two layers was an asymmetric membrane.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

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The invention claimed is:
 1. A method for a large-scale manufacture of asqualene-containing oil-in-water emulsion, comprising steps of: (i)forming providing a first emulsion having a first average oil dropletsize, wherein the first average oil droplet size is 5000 nm or lessand/or wherein the number of oil droplets having a size of >1.2 μm inthe first emulsion is 5×10¹¹/ml or less; (ii) microfluidizing the firstemulsion to form a second emulsion having a second average oil dropletsize which is less than the first average oil droplet size, wherein thenumber of oil droplets having a size of >1.2 μm in the second emulsionis 5×10¹⁰/ml or less; and (iii) filtrating the second emulsion using afirst hydrophilic polyethersulfone membrane layer having a poresize >0.3 μm and a second hydrophilic polyethersulfone membrane layerhaving a pore size <0.3 μm, thereby providing a squalene-containingoil-in-water emulsion, wherein at least one of the first and the secondhydrophilic polyethersulfone membrane layers is an asymmetric membrane,wherein the squalene-containing oil-in-water emulsion has a volume ofmore than or equal to 50 liters; and, (iv) filling the oil-in-wateremulsion into one or more sterile closed systems.
 2. The methodaccording to claim 1, wherein the second average oil droplet size is 500nm or less.
 3. The method according to claim 1, wherein the secondemulsion is prefiltered through a membrane prior to filtration.
 4. Themethod of claim 3, wherein the prefiltration and filtration steps arecarried out using a double-layer filter.
 5. The method of claim 3,wherein the filtration membrane, and optionally the prefiltrationmembrane, comprises a polymeric support material.
 6. The method of claim1, wherein the squalene-containing oil-in-water emulsion obtained afterstep (iii) is characterized in that the number of oil droplets having asize of >1.2 μm is 5×10⁸/ml or less.
 7. The method of claim 1, whereinthe filtering step (iii) comprises a double layer polyethersulfonemembrane.
 8. The method of claim 1, wherein the squalene-containingoil-in-water emulsion comprises, by volume, about 5% squalene, about0.5% polysorbate 80 and about 0.5% sorbitan trioleate.
 9. The method ofclaim 1, wherein the squalene-containing oil-in-water emulsion containsbetween 2-20% of oil by volume.
 10. The method of claim 1, wherein thesterile closed system is selected from the group consisting of: glassbottles, flexible, bags, vials and disposable syringes.
 11. A method fora large-scale manufacture of a squalene-containing oil-in-wateremulsion, comprising steps of: (i) subjecting a first emulsion having afirst average oil droplet size, wherein the first average oil dropletsize is 5000 nm or less and/or wherein the number of oil droplets havinga size of >1.2 μm in the first emulsion is 5×10¹¹/ml or less tomicrofluidization to form a second emulsion having a second average oildroplet size which is less than the first average oil droplet size,wherein the number of oil droplets having a size of >1.2 μm in thesecond emulsion is 5×10¹⁰/ml or less; (ii) filtrating the secondemulsion using a first hydrophilic polyethersulfone membrane layerhaving a pore size >0.3 μm and a second hydrophilic polyethersulfonemembrane layer having a pore size <0.3 μm, thereby providing asqualene-containing oil-in-water emulsion, wherein at least one of thefirst and the second hydrophilic polyethersulfone membrane layers is anasymmetric membrane wherein the squalene-containing oil-in-wateremulsion has a volume of more than or equal to 50 liters; and (iv)filling the oil-in-water emulsion into one or more sterile closedsystems.